RESUMO
Cre/lox site-specific recombination systems provide important tools for genetic manipulation. Here we present an efficient method for gene tagging and gene replacement using Cre recombinase-mediated cassette exchange (RMCE). The cassette consists of the S. pombe ura4(+) selectable marker flanked by a wild-type loxP site at one end and by a modified heterospecific lox site (loxM3) at the other. The cassette is stable because the flanking lox sites cannot recombine with each other. Following integration of the cassette at the chosen chromosomal locus, exchange is achieved by introducing a Cre-expression plasmid containing an equivalent cassette containing the required tag or gene sequence. Recombinants are selected by uracil prototrophy using the reagent 5-fluoroorotic acid (5-FOA). The cassette exchange system provides for repetitive integrations at the same locus, allowing different protein tags or gene sequences to be integrated quickly and efficiently. We have established a range of reagents and verified utility by C-terminally tagging the S. pombe rad4 and swi1 genes with yEGFP and the yEGFP derivatives yECFP and yECitrine and by transferring the coding sequence for both genes.
Assuntos
Marcação de Genes/métodos , Genes Fúngicos/genética , Schizosaccharomyces/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Fluorescência Verde/genética , Integrases/genética , Dados de Sequência Molecular , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Reação em Cadeia da Polimerase , Recombinação Genética , Schizosaccharomyces/efeitos dos fármacos , Proteínas de Schizosaccharomyces pombe/genética , Transglutaminases , Uracila/metabolismoRESUMO
FYSSION is a resource for researchers working on the fission yeast Schizosaccharomyces pombe. It currently comprises libraries of temperature-sensitive mutants in essential genes, and insertional mutants in non-essential genes, available for screening by visiting workers. Here we outline methods for constructing and using the libraries, and describe future prospects for functional genomics of this organism, here and elsewhere.
Assuntos
Genes Fúngicos/fisiologia , Biblioteca Genômica , Schizosaccharomyces/genética , Regulação Fúngica da Expressão Gênica , Genes Essenciais , Genômica , Modelos Biológicos , Mutagênese Insercional/fisiologia , Organismos Geneticamente Modificados , TemperaturaRESUMO
The fission yeast Schizosaccharomyces pombe grows in a single-celled form or can mate and undergo meiosis and sporulation. Here we show that wild-type S. pombe can also differentiate to form elaborately branched hyphae which invade deep into solid medium. Branches appear in the hyphae adjacent to unseparated septa. Electron microscopy reveals unusual multivesicular structures within the hyphae. Nitrogen deprivation appears to be the main stimulus for hyphal growth. No mitogen-activated protein kinase is necessary for the response. Inhibition of cyclic AMP (cAMP) production or signaling prevents the response, and exogenous cAMP promotes it, suggesting that detection of a good carbon source is required for hyphal growth but not for mating.